Andrea E. Schwarzbach

Rate of Lineage Origin Explains the Diversity Anomaly in the World’s Mangrove Vegetation

The contribution of nonecological factors to global patterns in diversity is evident when species richness differs between regions with similar habitats and geographic area. Mangrove environments in the Eastern Hemisphere harbor six times as many species of trees and shrubs as similar environments in the New World. Genetic divergence of mangrove lineages from terrestrial relatives, in combination with fossil evidence, suggests that mangrove diversity is limited by evolutionary transition into the stressful marine environment, the number of mangrove lineages has increased steadily over the Tertiary with little global extinction, and the diversity anomaly in mangrove vegetation reflects regional differences in the rate of origin of new mangrove lineages.

Phylogenetic position and floral function of Siparuna (Siparunaceae: Laurales)

Nucleotide sequences for two chloroplast genome regions, the rbcL gene and the trnL-trnF spacer region, were obtained for 22 genera and 30 species representing all major lineages of Laurales, with a special sampling effort being made in the phylogenetically problematic Monimiaceae sensu lato. Magnoliaceae, Winteraceae, Austrobaileyaceae, and Saururaceae were used as outgroups. A morphological character matrix for the same taxa was compiled and includes new floral-anatomical data for Siparunaceae, a lineage traditionally placed in Monimiaceae. Phylogenetic analyses of the molecular and morphological data based on maximum parsimony reveal that Siparuna and its sister taxon, the monotypic West African genus Glossocalyx, are not closely related to the remaining Monimiaceae, supporting the view that the Monimiaceae in the wide sense are polyphyletic. Based on morphology and chromosome numbers, Siparuna forms a clade with Atherospermataceae and Gomortegaceae, while based on rcbL sequences, it is in a clade with Hernandiaceae, Atherospermataceae, and Gomortegaceae. The trnL-trnF sequences provide no resolution for basal nodes in Laurales but agree with the rbcL and morphological analyses in strongly supporting a placement of Siparunaceae away from Monimiaceae s.str. Based on the phylogenetic hypotheses, we analyze some of the reported floral-morphological trends noted in Monimiaceae and Laurales, such as miniaturization of ovules, increasingly complete enclosure of reproductive organs, and aquisition of functional syncarpy. Both Siparuna and Glossocalyx have polycarpellate gynoecia embedded in massive receptacles covered by a membrane and uniovulate carpels with unitegmic ovules. The membrane, or floral roof, has a small, central pore for the styles or anthers to emerge at anthesis. It is more developed and thicker in female flowers than in male ones. Consequently, the styles in particular are forced into close physical contact. Flowers are pollinated by gall midges that oviposit into them through the pore in the floral roof, whereby they contact stamens or styles. Anatomical studies of flowers representing eight species show that styles fuse postgenitally at the height where they emerge through the pore, resulting in a joint transmission track for pollen tubes that originally landed on different stigmas. Lateral growth of pollen tubes, which results in switching between carpels, was observed in an experimentally pollinated species that had received large pollen loads.

Predominant and Substoichiometric Isomers of the Plastid Genome Coexist within Juniperus Plants and Have Shifted Multiple Times during Cupressophyte Evolution

Most land plant plastomes contain two copies of a large inverted repeat (IR) that promote high-frequency homologous recombination to generate isomeric genomic forms. Among conifer plastomes, this canonical IR is highly reduced in Pinaceae and completely lost from cupressophytes. However, both lineages have acquired short, novel IRs, some of which also exhibit recombinational activity to generate genomic structural diversity. This diversity has been shown to exist between, and occasionally within, cupressophyte species, but it is not known whether multiple genomic forms coexist within individual plants. To examine the recombinational potential of the novel cupressophyte IRs within individuals and between species, we sequenced the plastomes of four closely related species of Juniperus. The four plastomes have identical gene content and genome organization except for a large 36 kb inversion between approximately 250 bp IR containing trnQ-UUG. Southern blotting showed that different isomeric versions of the plastome predominate among individual junipers, whereas polymerase chain reaction and high-throughput read-pair mapping revealed the substoichiometric presence of the alternative isomeric form within each individual plant. Furthermore, our comparative genomic studies demonstrate that the predominant and substoichiometric arrangements of this IR have changed several times in other cupressophytes as well. These results provide compelling evidence for substoichiometric shifting of plastomic forms during cupressophyte evolution and suggest that substoichiometric shifting activity in plastid genomes may be adaptive.

Comparing DNA Extraction Methods for Analysis of Botanical Materials Found in Anti-Diabetic Supplements

A comparative performance evaluation of DNA extraction methods from anti-diabetic botanical supplements using various commercial kits was conducted, to determine which produces the best quality DNA suitable for PCR amplification, sequencing and species identification. All plant materials involved were of suboptimal quality showing various levels of degradation and therefore representing real conditions for testing herbal supplements. Eight different DNA extraction methods were used to isolate genomic DNA from 13 medicinal plant products. Two methods for evaluation, DNA concentration measurements that included absorbance ratios as well as PCR amplifiability, were used to determine quantity and quality of extracted DNA. We found that neither DNA concentrations nor commonly used UV absorbance ratio measurements at A260/A280 between 1.7 and 1.9 are suitable for globally predicting PCR success in these plant samples, and that PCR amplifiablity itself was the best indicator of extracted product quality. However, our results suggest that A260/A280 ratios below about 1.3 and above 2.3 indicated a DNA quality too poor to amplify. Therefore, A260/A280 measurements are not useful to identify samples that likely will amplify but can be used to exclude samples that likely will not amplify reducing the cost for unnecessarily subjecting samples to PCR. The two Nucleospin® plant II kit extraction methods produced the most pure and amplifiable genomic DNA extracts. Our results suggest that there are clear, discernable differences between extraction methods for low quality plant samples in terms of producing contamination-free, high-quality genomic DNA to be used for further analysis.

Apo B100 similarities to viral proteins suggest basis for LDL-DNA binding and transfection capacity

LDL mediates transfection with plasmid DNA in a variety of cell types in vitro and in several tissues in vivo in the rat. The transfection capacity of LDL is based on apo B100, as arginine/lysine clusters, suggestive of nucleic acid-binding domains and nuclear localization signal sequences, are present throughout the molecule. Apo E may also contribute to this capacity because of its similarity to the Dengue virus capsid proteins and its ability to bind DNA. Synthetic peptides representing two apo B100 regions with prominent Arg/Lys clusters were shown to bind DNA. Region 1 (0014Lys-Ser0160) shares sequence motifs present in DNA binding domains of Interferon Regulatory Factors and Flaviviridae capsid/core proteins. It also contains a close analog of the B/E receptor ligand of apo E. Region 1 peptides, B1-1 (0014Lys-Glu0054) and B1-2 (0055Leu-Ala0096), mediate transfection of HeLa cells but are cytotoxic. Region 2 (3313Asp-Thr3431), containing the known B/E receptor ligand, shares analog motifs with the human herpesvirus 5 immediate-early transcriptional regulator (UL122) and Flaviviridae NS3 helicases. Region 2 peptides, B2-1 (3313Asp-Glu3355), and B2-2 (3356Gly-Thr3431) are ineffective in cell transfection and are noncytotoxic. These results confirm the role of LDL as a natural transfection vector in vivo, a capacity imparted by the apo B100, and suggest a basis for Flaviviridae cell entry.